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6 Mice
Int. J. Radiation Oncology Biol. Phys., Vol. 69, No. 4, pp. 1272–1281, 2007 Copyright Ó 2007 Elsevier Inc. Printed in the USA. All rights reserved 0360-3016/07/$–see front matter
doi:10.1016/j.ijrobp.2007.07.2336
BIOLOGY CONTRIBUTION
IDENTIFICATION OF POSSIBLE CANDIDATE BIOMARKERS FOR LOCAL OR WHOLE BODY RADIATION EXPOSURE IN C57BL/6 MICE HAE-JUNE LEE, D.V.M., PH.D.,* MINYOUNG LEE, PH.D.,* CHANG-MO KANG, PH.D.,y DOOIL JEOUNG, PH.D.,z SANGWOO BAE, PH.D.,* CHUL-KOO CHO, M.D., PH.D.,x AND YUN-SIL LEE, PH.D.* * Laboratory of Radiation Effect, y Laboratory of Radiation Cytogenetics and Epidemiology, and x Department of Radiation Oncology, Korea Institute of Radiological and Medical Sciences, Seoul, Republic of Korea; and z Division of Life Sciences, Kangwon National University College of Natural Sciences, Chuncheon, Kangwon-Do, Republic of Korea Purpose: Specific genes expressed as a result of whole body exposure to g-radiation have been previously identified. In this study, we examined the genes further as possible biomarkers for the blood lymphocytes of C57BL/6 mice after whole body or local irradiation of the thorax, abdomen, and left subphrenic area. Methods and Materials: We performed reverse transcriptase-polymerase chain reaction and real-time reverse transcriptase-polymerase chain reaction analysis of genes encoding platelet membrane glycoprotein IIb, protein tyrosine kinase, sialyltransferase, and Cu/ZnSOD in blood lymphocytes, lung tissue, spleen, and intestines. The protein expression in blood lymphocytes was confirmed by Western blot analysis. Results: The expression of platelet membrane glycoprotein IIb, protein tyrosine kinase, sialyltransferase, and Cu/ ZnSOD was significantly greater after 3 days as a result of 1 Gy of whole body irradiation. Moreover, local irradiation to the thorax, abdomen, or left subphrenic area, which are frequently exposed to therapeutic radiation doses, showed a tendency toward radiation-induced increased expression of these genes in both the blood and the locally irradiated organs. Western blot analysis also corroborated these results. Conclusion: Platelet membrane glycoprotein IIb, protein tyrosine kinase, sialyltransferase, and Cu/ZnSOD might be candidates for biomarkers of radiation exposure. However, additional experiments are required to reveal the relationship between the expression levels and the prognostic effects after irradiation. Ó 2007 Elsevier Inc. Radiation exposure markers, Blood, Local exposure, Whole body exposure.
INTRODUCTION Exposure to ionizing radiation (IR) is a regular occurrence for humans. Although radiation is used in various technologies for life-saving purposes, through occupational, medical, environmental, accidental, and other sources, radiation exposure also presents a significant challenge to peoples’ health and psychological well-being. The conventional approach to the estimation of radiation exposure has been to integrate physical and clinical measurements to optimize dose assessment. However, these methods have practical limitations. Several attempts have been made to improve dosimetry using hematologic, biochemical, immunologic, and cytogenetic endpoints (1, 2). The lymphocyte count, which declines rapidly with radiation dose, is a sensitive indicator of exposure to irradiation (3, 4). However, because of the large variation in lymphocyte counts among normal individuals (5–8), it requires repeated
measurements for a prolonged period. Cytogenetic measurements of abnormalities in chromosome structure in metaphases of cultured lymphocytes (9, 10) have played an important role in the retrospective estimation of radiation dose received by Abomb survivors and individuals exposed during the Chernobyl nuclear accident (11, 12). Although the use of these cytogenetic techniques solves most of the problems inherent to physical dosimetry, issues of sensitivity still exist (13). Furthermore, a highly variable background level has been described in micronucleus assays, which is dependant on confounding factors, including gender, age, diet, and exposure to a wide range of environmental toxic compounds (14). Third, the analysis of dicentrics and micronuclei, with the exception of bone marrow, is time consuming and is applicable only to cell types that require culturing. Finally, these methods cannot determine the late effects of radiation exposure, which could be their most important defects as biomarkers.
Reprint requests to: Yun-Sil Lee, Ph.D., Laboratory of Radiation Effect, Korea Institute of Radiological and Medical Sciences, 215-4 Gongneung-Dong, Nowon-Ku, Seoul 139-706, Republic of Korea. Tel: (+82) 2-970-1325; Fax: (+82) 2-970-2402; E-mail: yslee@ kcch.re.kr Supported by the Korea Science and Engineering Foundation
(KOSEF) and the Ministry of Science and Technology (MOST) (Korean Government), through the National Nuclear Technology Program. Conflict of interest: none. Received March 27, 2007, and in revised form July 5, 2007. Accepted for publication July 6, 2007. 1272
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The new genomic tools identify the large-scale changes in gene expression profiles on the scale of minutes to hours after radiation exposure and have shown that a wide variety of cell process pathways and proteins are modified and activated (15–19). High-throughput gene expression profiling in human peripheral blood lymphocytes and other human cells or tissues, including irradiated skin, have identified several genes, including GADD45 and CDKN1A (1, 16, 20), BCL6, cytokines, and mitogen-activated protein kinase (21). The genes reported to be overexpressed in human peripheral blood lymphocytes (16) were analyzed in a previous study, and the radiation of inducible genes detected in the lung, spleen, and intestines were identified (22). In this study, we investigated whether these genes could be possible candidate blood biomarkers for radiation exposure, particularly after local exposure. METHODS AND MATERIALS Animals Female C57BL/6 mice (6–7 weeks old) were purchased from SLC (Hamamatsu, Japan) and kept at 22 2 C and 50% 10% humidity, with a 12-h light/12-h dark regimen. The Institutional Animal Care and Use Committee of the Korea Institute Radiological and Medical Science approved the studies, which were performed under the guidelines for the use and care of laboratory animals.
Irradiation Whole body irradiation (0.5, 1, and 2 Gy) was performed using a 137Cs g-ray source (Atomic Energy of Canada, Ontario, ON, Canada) at a dose rate of 3.51 Gy/min. Local region irradiation was performed with the mice anesthetized and localized field controlled irradiation using an irradiator (Theratron 780, Atomic Energy of Canada). The radiation field was 1.5 34 cm and the sourceto-skin distance was 80 cm. Groups of 9 mice were given 0.5, 1, and 2 Gy of radiation at a dose rate of 190 cGy/min on the surface of the thoracic, abdominal, and left subphrenic regions, per the experimental design. The local thoracic irradiation was a 1.5-cm-wide beam with the interior margin at the level of the xiphisternum. The abdomen was partially irradiated at 1 cm below the xiphisternum. The intestinal irradiation targeted most of the jejunum, ileum, and cecum and a part of the proximal colon. The center of the g-ray field was focused on the last rib of each mouse at the lateral surface for the right recumbent position, left subphrenic area, including the spleen. The control animals were sham-irradiated by placing them under anesthesia and placing them in the irradiator.
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TRIreagent for 10 min. The homogenized sample was centrifuged and the RNA extracted from the supernatant. The RNA sample was treated with DNase I, followed by a phenol/chloroform extraction. The blood samples that had been collected into PAXgene Blood RNA Tubes were gently mixed at room temperature on a hematology mixer for $2 h to ensure complete cell lysis and inactivation of ribonucleases before isolating the total RNA. Total RNA was also isolated according to the PAXgene protocol, and the quality of the isolated RNA was assayed by agarose gel electrophoresis by analyzing 18S and 28S RNA.
Reverse transcriptase-polymerase chain reaction For reverse transcriptase-polymerase chain reaction (RT-PCR), 0.5 mg aliquots of the total RNA from the spleen, lung, intestine, brain, and blood of the mice were used. The reaction mixture contained 2 mL of 10 RT buffer (Qiagen), 2 mL of 5 mM dNTPs (Qiagen), 1 mL of 10 U/mL RNAsin (Promega), 2 mL of 10 mM oligo (dT)-15 primer, 1.25 mg of total RNA, and 1 mL of Omniscript reverse transcriptase (Qiagen) at a final volume of 20 mL. The mixture was incubated at 37 C for 1 h, and the transcription reaction was quenched by heating the mixture at 95 C for 5 min, followed by annealing at 52 C for 1 min, and extension at 72 C for 30 s. Next, the reaction mixture was denatured for 35 cycles at 95 C for 30 s. The negative controls lacking RNA or RT were routinely included in the testing. The primer sequences used were as follows: sense 50 -ATTCAGCGCGTTACCATTTC-30 and antisense 50 -TGGCCC ACCTATGGAAAATA-30 for protein tyrosine kinase (636 bp); sense 50 -ACCGAGGTCACCTTTGACAC-30 and antisense 50 -T GCATCGGAAGTAAGCCTCT-30 for platelet membrane glycoprotein IIb (928 bp); sense 50 -CTGCCCAAGGAGAACTTCAG-30 and antisense 50 -ACACATCTGTCTTGCGCTTG-30 for sialyltransferase (533 bp); sense 50 -CGGTGAACCAGTTGTGTTGT-30 and antisense 50 -CACCTTTGCCCAAGTCATCT-30 for Cu/ ZnSOD (315 bp); sense 50 -AGGCTGTGCTGTCCCTGTATG-30 and antisense 50 -ACCCAAGAAGGAAGGCTGGAAA-30 for bactin (373 bp). The PCR products were analyzed with 2% agarose gel electrophoresis and stained with ethidium bromide. Next, the products were quantified using an image analyzer with the MCID software program (Image Research, Ontario, ON, Canada) and processed using Fluoro-S MultiImager (Bio-Rad Laboratories, Hercules, CA) for densitometric analysis.
Quantitative real-time PCR
At 1 and 3 days after irradiation, the subject mice were killed, followed by quickly harvesting tissue from the lung, spleen, intestine, and brain that were subsequently stored in liquid nitrogen. The peripheral blood samples were collected from the abdominal veins into tubes containing sodium citrate or into PAXgene Blood RNA Tubes (Qiagen). Also, the blood from 3 mice were pooled for each RNA or protein sample.
Real-time PCR analysis was performed using a DNA Engine2.OPTICON (MJ Research, Waltham, MA) and the LightCycler-FastStart DNA Master SYBR Green I mix (Roche, Basel, Switzerland). The reactions were performed in a final volume of 15 mL, which was adjusted to 4 mM of MgCl2 containing 500 nM of each primer and 2 mL of the DNA template. The real-time PCR cycling conditions were as follows: 94 C for 5 min, followed by 35 cycles for 1 min at 95 C, 1 min at 52 C, and 1 min at 72 C. Next, fluorescence measurement was performed at a polymerization temperature set at 68 C to facilitate accurate fluorescence measurements despite the low-melting point of the analyzed PCR products. After PCR analysis, a thermal melt profile was performed to identify the amplicon. To determine the threshold cycle (Ct), the fluorescence threshold level was set manually in the early phase of PCR amplification.
Total RNA isolation
Antibodies and immunoblot analysis
Total RNA was extracted from the tissues using TRIreagent (Molecular Research Center, Cincinnati, OH) according to the manufacturer’s protocol. Each 0.1-g sample was homogenized in 1 mL of
The protein level was detected using the mouse monoclonal CD41/integrin 2-a (Abcam, Cambridge, MA) for platelet membrane glycoprotein IIb and rabbit anti-Cu/Zn superoxide dismutase
Tissue preparation
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polyclonal antibody (StressGene, Victoria, BC, Canada) for Cu/ ZnSOD. Sialyltransferase was detected using a polyclonal rabbit antiserum that was raised against the GYLPKENFRTKAGP (amino acids 164–177 of mouse sialyltransferase) peptide. The organ tissues were homogenized with a PRO200 Homogenizer (PRO Scientific, Oxford, UK) in a lysis buffer (PRO-PREPTM, iNtRON, Gyeonggi-Do, Korea). The peripheral blood mononuclear cells were isolated using Histopaque (Sigma Chemical, St. Louis, MO) density gradient centrifugation, according to the manufacturer’s recommendations. The cell pellets were also placed in the lysis buffer. The protein concentration was determined using the Bradford method (Bio-Rad). For polyacrylamide gel electrophoresis and Western blotting, the proteins were dissolved in a lysis buffer (120 mM NaCl, 40 mM Tris [pH 8.0], 0.1% NP-40), and the samples were boiled for 5 min. Equal amounts of protein (40 mg/well) were then analyzed on 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis. After electrophoresis, the proteins were transferred onto a nitrocellulose membrane and processed for immunoblotting. Blocking was performed in an incubation solution (5% nonfat dry milk in phosphate-buffered saline and 0.1% Tween-20) for 2 h at room temperature. After blocking, the membranes were probed with the corresponding antibody for 18 h at 4 C, followed by washing with 5% nonfat dry milk in phosphate-buffered saline and 0.1% Tween-20. The blots were then incubated with horseradish peroxidase-conjugated secondary antibody, diluted at 1:5,000, and the specific bands were visualized by enhanced chemiluminescence (Amersham International, Buckinghamshire, UK). The autoradiographs were recorded on X-Omat Duplicating film (Eastman Kodak, Rochester, NY). The densitometric analysis of the Western blot was performed using the Fluoro-S MultiImager (Bio-Rad).
Statistical analysis The data are expressed as the mean standard deviation, and statistical significance was determined using Student’s t test for comparison between the mean values. The significance of the differences for all the groups was calculated using one-way analysis of variance with a post-hoc Tukey test. The statistical significance was set a priori at p < 0.05. The relationship between the dose dependence of protein expression and the local irradiation region was tested with a linear regression model (23). All tests were performed using the Statistical Package for Social Sciences, version 14.0, software (SPSS, Chicago, IL).
RESULTS Identification of genes responsible for radiation exposure in blood lymphocytes In our previous study, using whole body irradiation, the platelet membrane glycoprotein IIb, protein tyrosine kinase, sialyltransferase, and Cu/ZnSOD were radiation-responsive genes detected in the lung, intestine, and spleen of C57BL/ 6 mice. However, these genes were not found in the heart or brain, suggesting that these genes are radiation sensitive after irradiating the lung, intestine, and spleen (22). The present study examined the expression of these genes in the blood lymphocytes of C57BL/6 mice after 1 Gy of whole body radiation. The RT-PCR analysis indicated that the expression of these genes increased after 1 day of radiation and continued to increase for a total of 3 days (Fig. 1A). A significant increase was only observed after the third day of radiation (4.3-, 3.6-, 2.9-, and 3.1-fold increase, respectively) in
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mRNA expression of platelet membrane glycoprotein IIb, protein tyrosine kinase, sialyltransferase, and Cu/ZnSOD, respectively, compared with normal nonirradiated controls. The quantification of expression, using real-time RT-PCR analysis in the blood lymphocytes also confirmed the significant increase in expression of the four genes on the third day of 1 Gy whole body radiation (5.49, 3.83, 5.36, and 4.45-fold increase in mRNA expression of platelet membrane glycoprotein IIb, protein tyrosine kinase, sialyltransferase, and Cu/ZnSOD, respectively; Fig. 1B). Increased gene expression of radiation-responsive genes in blood lymphocytes of locally irradiated mice The detection of the radiation-responsive genes, platelet membrane glycoprotein IIb, protein tyrosine kinase, sialyltransferase, and Cu/ZnSOD, was performed after local irradiation of 1 Gy to the thorax, abdomen, and left subphrenic area. These areas are frequently exposed to therapeutic or diagnostic radiation. On the third day after radiation, which had showed the greatest expression of these genes in the blood lymphocytes after 1 Gy whole body radiation (Fig. 1B), RT-PCR analysis suggested that these genes had a tendency for overexpression in each of the irradiated organs (i.e., lung, intestine, and spleen) after local irradiation, although statistical significance was not observed in all cases. The brain, which had not been irradiated, showed no alterations in the expression of these genes (Fig. 2A), suggesting that these genes were overexpressed only in the irradiated organs. RT-PCR analysis using blood lymphocytes at 1 or 3 days after local irradiation to the thorax, abdomen, or left subphrenic area revealed a significant increase in mRNA expression of the platelet membrane glycoprotein IIb (3.5- and 3.6-fold increases in the lung and spleen, respectively), protein tyrosine kinase (3.7- and 3.0-fold increases in the lung and spleen, respectively), sialyltransferase (2.3- and 2.4-fold increases in the lung and spleen, respectively), and Cu/ZnSOD (2.3and 3.5-fold increases in the lung and spleen, respectively). This was observed only after the third day of radiation. An increase in gene expression was observed in the intestines. A significant difference was observed in platelet membrane glycoprotein IIb (2.0- and 3.0-fold increases at the first and third days after irradiation, respectively), sialyltransferase (1.9-fold increase the first day after irradiation), and Cu/ ZnSOD (2.0-fold increase the third day after irradiation; Fig. 2B), suggesting that these genes are possible candidates as markers of radiation exposure after local irradiation to the thorax, abdomen, and left subphrenic area, even though the expression time kinetics was different. The quantification of expression by real-time RT-PCR also indicated that local irradiation significantly increased the gene expression of the platelet membrane glycoprotein IIb (6.02-, 5.95-, and 2.27-fold increase in the lung, intestine, and spleen, respectively), protein tyrosine kinase (8.19-, 4.60-, and 3.04-fold increase in the lung, intestine, and spleen, respectively), sialyltransferase (6.87-, 1.26-, and 2.18-fold increase in the lung, intestine, and spleen, respectively), and Cu/ZnSOD (2.31-, 3.03-, and 1.93-fold increase in the lung, intestine,
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Fig. 1. mRNA expression level of genes encoding protein tyrosine kinase, platelet membrane glycoprotein IIb, sialyltransferase, and Cu/ZnSOD in peripheral blood after 1 Gy of whole body irradiation (IR). After 1 and 3 days of 1 Gy IR, peripheral blood lymphocytes were isolated, and total RNA was prepared and analyzed by formaldehyde/agarose gel electrophoresis, followed by transfer to a nylon membrane as described in ‘‘Methods and Materials’’ section. (A) Mean standard deviation of induction calculated as ratio of non-IR normal control (NC) mice. (B) Real-time reverse transcriptasepolymerase chain reaction analysis for these four genes also performed. Peripheral blood lymphocytes from 3 mice pooled for each RNA isolation. Bars represent mean of measurements from three experiments. *Statistical significance from NC mice set a priori at p < 0.05. Data depicted represent two independent experiment sets.
and spleen, respectively) in blood lymphocytes the third day after irradiation (Fig. 2C). In general, a significant increase in these genes was expressed primarily on the third day after irradiation, rather than the first day after irradiation. Protein expression of radiation-responsive genes in blood lymphocytes of whole body or locally irradiated mice The detection of protein is better for the development of biomarkers than mRNA. Therefore, this study examined whether a relationship existed between mRNA expression and protein expression. One Gray of whole body or local irradiation significantly increased the protein levels of the platelet membrane glycoprotein IIb, sialyltransferase, and Cu/ZnSOD in each organ at 1 or 3 days after irradiation (Fig. 3A, B). In addition, the effects were more pronounced for local irradiation. The sialyltransferase antibody sometimes detected both the insoluble (48 kDa) and soluble (37–42 kDa) forms, and irradiation increased induction of the soluble form. The protein level of protein tyrosine kinase
was not detected because no antibody is commercially available. The protein expression patterns after 0.5, 1, and 2 Gy of radiation were examined at the third day of radiation to determine whether the protein expression after local or whole body irradiation was dependent on the radiation dose. We chose the third day because the effects were more evident than on the first day. The protein expression level in the blood lymphocytes after local irradiation to the thorax, abdomen, and left subphrenic area was increased; however, local thoracic irradiation significantly increased only platelet membrane glycoprotein IIb and Cu/ZnSOD as a function of dose when analyzed with a linear regression model. Sialyltransferase expression was also dose dependent but only when exposed to the left subphrenic area (Fig. 3C and Table 1). DISCUSSION We anticipate continued development of new technologies and procedures in the detection of radiation exposure. Moreover, these developments will continue to fuel the increased
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Fig. 2. mRNA expression of protein tyrosine kinase, platelet membrane glycoprotein IIb, sialyltransferase, and Cu/ZnSOD in peripheral blood lymphocytes or organs exposed to localized irradiation (IR). (A) Reverse transcriptase-polymerase chain reaction (RT-PCR) analysis of four genes in intestine, lung, and spleen tissue from 3 mice exposed to local 1 Gy IR and compared with gene expression in brain 3 days after IR. (B) Real-time RT-PCR or quantification for real-time RT-PCR. (C) Analysis for four genes also performed in peripheral blood lymphocytes after 1 Gy of local IR to thorax, abdomen, and left subphrenic area. Peripheral blood lymphocytes from 3 mice pooled for each RNA isolation. Bars represent mean of measurements from three experiments. Mean standard deviation calculated as ratio of nonirradiated normal control (NC) mice. *Statistical significance from NC mice set a priori at p < 0.05. Representative data of two independent experiment sets shown. Figure is continued on next page.
use of radiation technology in industry and medical diagnosis and therapy. From the experience with radiation accidents, it is evident that biologic indicators are needed to obtain enough information of the distribution and extent of radiation exposure. These biologic indicators could have an additional advantage because the radiation damage to an individual could be measured, including the variability of an individual’s radiosensitivity. Of the many biologic parameters that have been studied, chromosomal damage in lymphocytes is the most promising (24–26). Different cytogenetic changes (i.e., increased frequency of chromosomal aberrations and micronuclei), are well known in individuals exposed to ionizing radiation (27–30). However, this method is time consuming, and the recently developed large-scale gene expression showed that the individual prognosis after
radiation exposure could be estimated by determining their function. The present study has identified candidate biomarkers for radiation exposure that could be detected in blood samples of whole body or locally irradiated mice. In a previous study, five organs, including the intestine, spleen, lung, heart, and brain, were studied after 0.2 Gy of radiation exposure. RTPCR was applied to the 23 genes that have been identified by cDNA microarray analysis to show increased expression in the peripheral blood lymphocytes after 1 Gy of radiation (16). Of these, the platelet membrane glycoprotein IIb, protein tyrosine kinase, sialyltransferase, and Cu/ZnSOD were expressed in the intestine, lung, or spleen but not in the heart or brain (22), suggesting these as possible gene candidates for measuring exposure to the spleen, lung, or intestines for these
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Fig. 2. (Continued)
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Fig. 3. Protein expression in organ tissues or peripheral blood lymphocytes exposed to whole body or local irradiation (IR). After mice were exposed to 1 Gy of (A) whole body or (B) local IR, protein expression of platelet membrane glycoprotein IIb, sialyltransferase, and Cu/ZnSOD in intestine, lung, and spleen were examined at 1 and 3 days after IR. Peripheral blood lymphocytes or organs from 3 mice pooled for RNA isolation. Bars depict results from three experiments. Mean standard deviation calculated as ratio of non-IR normal control (NC) mice. *Statistical significance from NC mice set a priori at p < 0.05. Representative data of two independent experiment sets shown. (C) Ratio of peripheral blood lymphocytes isolated at 3 days after local IR to thorax, abdomen, and left subphrenic area. Pattern of protein expression of platelet membrane glycoprotein IIb, sialyltransferase, and Cu/ZnSOD examined, and peripheral blood lymphocytes from 3 mice pooled for each protein extraction, and two experiments performed for each dose. Representative data depicted as two independent experimental data sets. Figure is continued on next page.
radiation-sensitive organs. Sialyltransferase is a glycosphingolipid synthase that undergoes characteristic changes during an oncogenic transformation (30) and has influenza virus sensitivity (31). Cu/ZnSOD is a protein tyrosine kinase and platelet membrane glycoprotein IIb has functions that are more related to cellular resistance than to cell death, suggesting that the radiation exposure biomarker might not be related to function. When mRNA expression of the platelet membrane glycoprotein IIb, protein tyrosine kinase, sialyltransferase, and Cu/ZnSOD was examined in the blood after whole body irradiation, 1 Gy of radiation was found to induce these genes (Fig. 1). Similarly, local irradiation to the thorax, abdomen, or left subphrenic area also increased the mRNA levels of these genes in the irradiated lung, spleen, and intestine and blood samples (Fig. 2), suggesting that these genes might be candidates for biomarkers of radiation exposure. The most important finding of this study was the identification of possible protein biomarkers for radiation exposure, because proteins would be more suitable as biomarkers. Moreover, the changes in the expression levels of the proteins
were more pronounced compared with the changes in mRNA expression in the organs of locally irradiated mice. Therefore, the radiation induced post-translational modification of these proteins or the protein itself is more sensitive to the detection of radiation exposure. However, radiation did not induce the expression of these proteins in the blood lymphocytes after whole body irradiation. Rather, whole body irradiation reduced the protein expression (data not shown), suggesting that some proteolytic degradation in blood samples might be activated by radiation. When the protein expression in the blood lymphocytes of the locally irradiated mice was examined, no proteolytic degradation of these proteins could be detected, suggesting that local irradiation has less effect on the proteolytic degradation pathways in blood lymphocytes (the blood might be more affected by proteolytic degradation after radiation). Therefore, mRNA detection in blood samples appears to be more suitable after whole body radiation for which the detection of these proteins failed as biomarkers. Despite this, protein expression appears to be more suitable in the case of local irradiation (especially in lung tissue).
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Fig. 3. (Continued)
Table 1. Regression analysis of radiation dose dependence for protein expressions in peripheral blood lymphocytes after 1 Gy of local irradiation Thorax
Abdomen
Left subphrenic area
Irradiated region
b1*
p
b2*
p
b3*
p
Platelet membrane glycoprotein IIb Cu/ZnSOD Sialyltransferase
0.863
0.004
0.355
0.623
0.038
0.812
0.454 0.470
0.001 0.060
0.350 0.716
0.053 0.072
0.305 2.108
0.074 0.029
* Regression coefficient.
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We do not know exactly why the genes of platelet membrane glycoprotein IIb, protein tyrosine kinase, sialyltransferase, and Cu/ZnSOD could be detected in the blood lymphocytes when locally irradiated at the thorax, abdomen, and left subphrenic area. One possibility is that the lymphocytes inside the local irradiated organs might have been affected by the radiation and they were detected in the blood. Another possibility is that cytokines and soluble factors might be secreted as a result of radiation in locally irradiated organs and they, in turn, might affect the lymphocytes. Gene changes in the lymphocytes were more pronounced at 3 days (as opposed to 1 day) of radiation exposure, suggesting that the gene expression of lymphocytes after local irradiation might be an indirect consequence of radiation exposure. One of the important features of biomarkers is dose-dependent expression. Therefore, several radiation doses were used to determine whether the change in the expression of these proteins is dependent. Local radiation doses of 0.5, 1, and 2
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Gy to the thorax, abdomen, and left subphrenic area induced protein expression of platelet membrane glycoprotein IIb, sialyltransferase, and Cu/ZnSOD in blood lymphocytes; however, dose dependency was only observed for platelet membrane glycoprotein IIb and Cu/ZnSOD after thoracic irradiation and sialyltransferase after irradiation to the left subphrenic area. These results suggest possible candidates for biodosimetry, because even the 0.5-Gy radiation dose induced protein expression. In addition, it is possible that these proteins could also be candidate markers for low-dose local radiation to the thorax, abdomen, or left subphrenic area. Moreover, these proteins might be valuable biomarkers for predicting early and mechanism-specific radiation exposure, in addition to revealing the future effect of radiation earlier than with conventional methods (particularly after local radiation exposure), even though more detailed experiments are needed for the establishment of these proteins as biomarkers of radiation exposure.
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doi:10.1016/j.ijrobp.2007.07.2336
BIOLOGY CONTRIBUTION
IDENTIFICATION OF POSSIBLE CANDIDATE BIOMARKERS FOR LOCAL OR WHOLE BODY RADIATION EXPOSURE IN C57BL/6 MICE HAE-JUNE LEE, D.V.M., PH.D.,* MINYOUNG LEE, PH.D.,* CHANG-MO KANG, PH.D.,y DOOIL JEOUNG, PH.D.,z SANGWOO BAE, PH.D.,* CHUL-KOO CHO, M.D., PH.D.,x AND YUN-SIL LEE, PH.D.* * Laboratory of Radiation Effect, y Laboratory of Radiation Cytogenetics and Epidemiology, and x Department of Radiation Oncology, Korea Institute of Radiological and Medical Sciences, Seoul, Republic of Korea; and z Division of Life Sciences, Kangwon National University College of Natural Sciences, Chuncheon, Kangwon-Do, Republic of Korea Purpose: Specific genes expressed as a result of whole body exposure to g-radiation have been previously identified. In this study, we examined the genes further as possible biomarkers for the blood lymphocytes of C57BL/6 mice after whole body or local irradiation of the thorax, abdomen, and left subphrenic area. Methods and Materials: We performed reverse transcriptase-polymerase chain reaction and real-time reverse transcriptase-polymerase chain reaction analysis of genes encoding platelet membrane glycoprotein IIb, protein tyrosine kinase, sialyltransferase, and Cu/ZnSOD in blood lymphocytes, lung tissue, spleen, and intestines. The protein expression in blood lymphocytes was confirmed by Western blot analysis. Results: The expression of platelet membrane glycoprotein IIb, protein tyrosine kinase, sialyltransferase, and Cu/ ZnSOD was significantly greater after 3 days as a result of 1 Gy of whole body irradiation. Moreover, local irradiation to the thorax, abdomen, or left subphrenic area, which are frequently exposed to therapeutic radiation doses, showed a tendency toward radiation-induced increased expression of these genes in both the blood and the locally irradiated organs. Western blot analysis also corroborated these results. Conclusion: Platelet membrane glycoprotein IIb, protein tyrosine kinase, sialyltransferase, and Cu/ZnSOD might be candidates for biomarkers of radiation exposure. However, additional experiments are required to reveal the relationship between the expression levels and the prognostic effects after irradiation. Ó 2007 Elsevier Inc. Radiation exposure markers, Blood, Local exposure, Whole body exposure.
INTRODUCTION Exposure to ionizing radiation (IR) is a regular occurrence for humans. Although radiation is used in various technologies for life-saving purposes, through occupational, medical, environmental, accidental, and other sources, radiation exposure also presents a significant challenge to peoples’ health and psychological well-being. The conventional approach to the estimation of radiation exposure has been to integrate physical and clinical measurements to optimize dose assessment. However, these methods have practical limitations. Several attempts have been made to improve dosimetry using hematologic, biochemical, immunologic, and cytogenetic endpoints (1, 2). The lymphocyte count, which declines rapidly with radiation dose, is a sensitive indicator of exposure to irradiation (3, 4). However, because of the large variation in lymphocyte counts among normal individuals (5–8), it requires repeated
measurements for a prolonged period. Cytogenetic measurements of abnormalities in chromosome structure in metaphases of cultured lymphocytes (9, 10) have played an important role in the retrospective estimation of radiation dose received by Abomb survivors and individuals exposed during the Chernobyl nuclear accident (11, 12). Although the use of these cytogenetic techniques solves most of the problems inherent to physical dosimetry, issues of sensitivity still exist (13). Furthermore, a highly variable background level has been described in micronucleus assays, which is dependant on confounding factors, including gender, age, diet, and exposure to a wide range of environmental toxic compounds (14). Third, the analysis of dicentrics and micronuclei, with the exception of bone marrow, is time consuming and is applicable only to cell types that require culturing. Finally, these methods cannot determine the late effects of radiation exposure, which could be their most important defects as biomarkers.
Reprint requests to: Yun-Sil Lee, Ph.D., Laboratory of Radiation Effect, Korea Institute of Radiological and Medical Sciences, 215-4 Gongneung-Dong, Nowon-Ku, Seoul 139-706, Republic of Korea. Tel: (+82) 2-970-1325; Fax: (+82) 2-970-2402; E-mail: yslee@ kcch.re.kr Supported by the Korea Science and Engineering Foundation
(KOSEF) and the Ministry of Science and Technology (MOST) (Korean Government), through the National Nuclear Technology Program. Conflict of interest: none. Received March 27, 2007, and in revised form July 5, 2007. Accepted for publication July 6, 2007. 1272
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The new genomic tools identify the large-scale changes in gene expression profiles on the scale of minutes to hours after radiation exposure and have shown that a wide variety of cell process pathways and proteins are modified and activated (15–19). High-throughput gene expression profiling in human peripheral blood lymphocytes and other human cells or tissues, including irradiated skin, have identified several genes, including GADD45 and CDKN1A (1, 16, 20), BCL6, cytokines, and mitogen-activated protein kinase (21). The genes reported to be overexpressed in human peripheral blood lymphocytes (16) were analyzed in a previous study, and the radiation of inducible genes detected in the lung, spleen, and intestines were identified (22). In this study, we investigated whether these genes could be possible candidate blood biomarkers for radiation exposure, particularly after local exposure. METHODS AND MATERIALS Animals Female C57BL/6 mice (6–7 weeks old) were purchased from SLC (Hamamatsu, Japan) and kept at 22 2 C and 50% 10% humidity, with a 12-h light/12-h dark regimen. The Institutional Animal Care and Use Committee of the Korea Institute Radiological and Medical Science approved the studies, which were performed under the guidelines for the use and care of laboratory animals.
Irradiation Whole body irradiation (0.5, 1, and 2 Gy) was performed using a 137Cs g-ray source (Atomic Energy of Canada, Ontario, ON, Canada) at a dose rate of 3.51 Gy/min. Local region irradiation was performed with the mice anesthetized and localized field controlled irradiation using an irradiator (Theratron 780, Atomic Energy of Canada). The radiation field was 1.5 34 cm and the sourceto-skin distance was 80 cm. Groups of 9 mice were given 0.5, 1, and 2 Gy of radiation at a dose rate of 190 cGy/min on the surface of the thoracic, abdominal, and left subphrenic regions, per the experimental design. The local thoracic irradiation was a 1.5-cm-wide beam with the interior margin at the level of the xiphisternum. The abdomen was partially irradiated at 1 cm below the xiphisternum. The intestinal irradiation targeted most of the jejunum, ileum, and cecum and a part of the proximal colon. The center of the g-ray field was focused on the last rib of each mouse at the lateral surface for the right recumbent position, left subphrenic area, including the spleen. The control animals were sham-irradiated by placing them under anesthesia and placing them in the irradiator.
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TRIreagent for 10 min. The homogenized sample was centrifuged and the RNA extracted from the supernatant. The RNA sample was treated with DNase I, followed by a phenol/chloroform extraction. The blood samples that had been collected into PAXgene Blood RNA Tubes were gently mixed at room temperature on a hematology mixer for $2 h to ensure complete cell lysis and inactivation of ribonucleases before isolating the total RNA. Total RNA was also isolated according to the PAXgene protocol, and the quality of the isolated RNA was assayed by agarose gel electrophoresis by analyzing 18S and 28S RNA.
Reverse transcriptase-polymerase chain reaction For reverse transcriptase-polymerase chain reaction (RT-PCR), 0.5 mg aliquots of the total RNA from the spleen, lung, intestine, brain, and blood of the mice were used. The reaction mixture contained 2 mL of 10 RT buffer (Qiagen), 2 mL of 5 mM dNTPs (Qiagen), 1 mL of 10 U/mL RNAsin (Promega), 2 mL of 10 mM oligo (dT)-15 primer, 1.25 mg of total RNA, and 1 mL of Omniscript reverse transcriptase (Qiagen) at a final volume of 20 mL. The mixture was incubated at 37 C for 1 h, and the transcription reaction was quenched by heating the mixture at 95 C for 5 min, followed by annealing at 52 C for 1 min, and extension at 72 C for 30 s. Next, the reaction mixture was denatured for 35 cycles at 95 C for 30 s. The negative controls lacking RNA or RT were routinely included in the testing. The primer sequences used were as follows: sense 50 -ATTCAGCGCGTTACCATTTC-30 and antisense 50 -TGGCCC ACCTATGGAAAATA-30 for protein tyrosine kinase (636 bp); sense 50 -ACCGAGGTCACCTTTGACAC-30 and antisense 50 -T GCATCGGAAGTAAGCCTCT-30 for platelet membrane glycoprotein IIb (928 bp); sense 50 -CTGCCCAAGGAGAACTTCAG-30 and antisense 50 -ACACATCTGTCTTGCGCTTG-30 for sialyltransferase (533 bp); sense 50 -CGGTGAACCAGTTGTGTTGT-30 and antisense 50 -CACCTTTGCCCAAGTCATCT-30 for Cu/ ZnSOD (315 bp); sense 50 -AGGCTGTGCTGTCCCTGTATG-30 and antisense 50 -ACCCAAGAAGGAAGGCTGGAAA-30 for bactin (373 bp). The PCR products were analyzed with 2% agarose gel electrophoresis and stained with ethidium bromide. Next, the products were quantified using an image analyzer with the MCID software program (Image Research, Ontario, ON, Canada) and processed using Fluoro-S MultiImager (Bio-Rad Laboratories, Hercules, CA) for densitometric analysis.
Quantitative real-time PCR
At 1 and 3 days after irradiation, the subject mice were killed, followed by quickly harvesting tissue from the lung, spleen, intestine, and brain that were subsequently stored in liquid nitrogen. The peripheral blood samples were collected from the abdominal veins into tubes containing sodium citrate or into PAXgene Blood RNA Tubes (Qiagen). Also, the blood from 3 mice were pooled for each RNA or protein sample.
Real-time PCR analysis was performed using a DNA Engine2.OPTICON (MJ Research, Waltham, MA) and the LightCycler-FastStart DNA Master SYBR Green I mix (Roche, Basel, Switzerland). The reactions were performed in a final volume of 15 mL, which was adjusted to 4 mM of MgCl2 containing 500 nM of each primer and 2 mL of the DNA template. The real-time PCR cycling conditions were as follows: 94 C for 5 min, followed by 35 cycles for 1 min at 95 C, 1 min at 52 C, and 1 min at 72 C. Next, fluorescence measurement was performed at a polymerization temperature set at 68 C to facilitate accurate fluorescence measurements despite the low-melting point of the analyzed PCR products. After PCR analysis, a thermal melt profile was performed to identify the amplicon. To determine the threshold cycle (Ct), the fluorescence threshold level was set manually in the early phase of PCR amplification.
Total RNA isolation
Antibodies and immunoblot analysis
Total RNA was extracted from the tissues using TRIreagent (Molecular Research Center, Cincinnati, OH) according to the manufacturer’s protocol. Each 0.1-g sample was homogenized in 1 mL of
The protein level was detected using the mouse monoclonal CD41/integrin 2-a (Abcam, Cambridge, MA) for platelet membrane glycoprotein IIb and rabbit anti-Cu/Zn superoxide dismutase
Tissue preparation
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polyclonal antibody (StressGene, Victoria, BC, Canada) for Cu/ ZnSOD. Sialyltransferase was detected using a polyclonal rabbit antiserum that was raised against the GYLPKENFRTKAGP (amino acids 164–177 of mouse sialyltransferase) peptide. The organ tissues were homogenized with a PRO200 Homogenizer (PRO Scientific, Oxford, UK) in a lysis buffer (PRO-PREPTM, iNtRON, Gyeonggi-Do, Korea). The peripheral blood mononuclear cells were isolated using Histopaque (Sigma Chemical, St. Louis, MO) density gradient centrifugation, according to the manufacturer’s recommendations. The cell pellets were also placed in the lysis buffer. The protein concentration was determined using the Bradford method (Bio-Rad). For polyacrylamide gel electrophoresis and Western blotting, the proteins were dissolved in a lysis buffer (120 mM NaCl, 40 mM Tris [pH 8.0], 0.1% NP-40), and the samples were boiled for 5 min. Equal amounts of protein (40 mg/well) were then analyzed on 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis. After electrophoresis, the proteins were transferred onto a nitrocellulose membrane and processed for immunoblotting. Blocking was performed in an incubation solution (5% nonfat dry milk in phosphate-buffered saline and 0.1% Tween-20) for 2 h at room temperature. After blocking, the membranes were probed with the corresponding antibody for 18 h at 4 C, followed by washing with 5% nonfat dry milk in phosphate-buffered saline and 0.1% Tween-20. The blots were then incubated with horseradish peroxidase-conjugated secondary antibody, diluted at 1:5,000, and the specific bands were visualized by enhanced chemiluminescence (Amersham International, Buckinghamshire, UK). The autoradiographs were recorded on X-Omat Duplicating film (Eastman Kodak, Rochester, NY). The densitometric analysis of the Western blot was performed using the Fluoro-S MultiImager (Bio-Rad).
Statistical analysis The data are expressed as the mean standard deviation, and statistical significance was determined using Student’s t test for comparison between the mean values. The significance of the differences for all the groups was calculated using one-way analysis of variance with a post-hoc Tukey test. The statistical significance was set a priori at p < 0.05. The relationship between the dose dependence of protein expression and the local irradiation region was tested with a linear regression model (23). All tests were performed using the Statistical Package for Social Sciences, version 14.0, software (SPSS, Chicago, IL).
RESULTS Identification of genes responsible for radiation exposure in blood lymphocytes In our previous study, using whole body irradiation, the platelet membrane glycoprotein IIb, protein tyrosine kinase, sialyltransferase, and Cu/ZnSOD were radiation-responsive genes detected in the lung, intestine, and spleen of C57BL/ 6 mice. However, these genes were not found in the heart or brain, suggesting that these genes are radiation sensitive after irradiating the lung, intestine, and spleen (22). The present study examined the expression of these genes in the blood lymphocytes of C57BL/6 mice after 1 Gy of whole body radiation. The RT-PCR analysis indicated that the expression of these genes increased after 1 day of radiation and continued to increase for a total of 3 days (Fig. 1A). A significant increase was only observed after the third day of radiation (4.3-, 3.6-, 2.9-, and 3.1-fold increase, respectively) in
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mRNA expression of platelet membrane glycoprotein IIb, protein tyrosine kinase, sialyltransferase, and Cu/ZnSOD, respectively, compared with normal nonirradiated controls. The quantification of expression, using real-time RT-PCR analysis in the blood lymphocytes also confirmed the significant increase in expression of the four genes on the third day of 1 Gy whole body radiation (5.49, 3.83, 5.36, and 4.45-fold increase in mRNA expression of platelet membrane glycoprotein IIb, protein tyrosine kinase, sialyltransferase, and Cu/ZnSOD, respectively; Fig. 1B). Increased gene expression of radiation-responsive genes in blood lymphocytes of locally irradiated mice The detection of the radiation-responsive genes, platelet membrane glycoprotein IIb, protein tyrosine kinase, sialyltransferase, and Cu/ZnSOD, was performed after local irradiation of 1 Gy to the thorax, abdomen, and left subphrenic area. These areas are frequently exposed to therapeutic or diagnostic radiation. On the third day after radiation, which had showed the greatest expression of these genes in the blood lymphocytes after 1 Gy whole body radiation (Fig. 1B), RT-PCR analysis suggested that these genes had a tendency for overexpression in each of the irradiated organs (i.e., lung, intestine, and spleen) after local irradiation, although statistical significance was not observed in all cases. The brain, which had not been irradiated, showed no alterations in the expression of these genes (Fig. 2A), suggesting that these genes were overexpressed only in the irradiated organs. RT-PCR analysis using blood lymphocytes at 1 or 3 days after local irradiation to the thorax, abdomen, or left subphrenic area revealed a significant increase in mRNA expression of the platelet membrane glycoprotein IIb (3.5- and 3.6-fold increases in the lung and spleen, respectively), protein tyrosine kinase (3.7- and 3.0-fold increases in the lung and spleen, respectively), sialyltransferase (2.3- and 2.4-fold increases in the lung and spleen, respectively), and Cu/ZnSOD (2.3and 3.5-fold increases in the lung and spleen, respectively). This was observed only after the third day of radiation. An increase in gene expression was observed in the intestines. A significant difference was observed in platelet membrane glycoprotein IIb (2.0- and 3.0-fold increases at the first and third days after irradiation, respectively), sialyltransferase (1.9-fold increase the first day after irradiation), and Cu/ ZnSOD (2.0-fold increase the third day after irradiation; Fig. 2B), suggesting that these genes are possible candidates as markers of radiation exposure after local irradiation to the thorax, abdomen, and left subphrenic area, even though the expression time kinetics was different. The quantification of expression by real-time RT-PCR also indicated that local irradiation significantly increased the gene expression of the platelet membrane glycoprotein IIb (6.02-, 5.95-, and 2.27-fold increase in the lung, intestine, and spleen, respectively), protein tyrosine kinase (8.19-, 4.60-, and 3.04-fold increase in the lung, intestine, and spleen, respectively), sialyltransferase (6.87-, 1.26-, and 2.18-fold increase in the lung, intestine, and spleen, respectively), and Cu/ZnSOD (2.31-, 3.03-, and 1.93-fold increase in the lung, intestine,
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Fig. 1. mRNA expression level of genes encoding protein tyrosine kinase, platelet membrane glycoprotein IIb, sialyltransferase, and Cu/ZnSOD in peripheral blood after 1 Gy of whole body irradiation (IR). After 1 and 3 days of 1 Gy IR, peripheral blood lymphocytes were isolated, and total RNA was prepared and analyzed by formaldehyde/agarose gel electrophoresis, followed by transfer to a nylon membrane as described in ‘‘Methods and Materials’’ section. (A) Mean standard deviation of induction calculated as ratio of non-IR normal control (NC) mice. (B) Real-time reverse transcriptasepolymerase chain reaction analysis for these four genes also performed. Peripheral blood lymphocytes from 3 mice pooled for each RNA isolation. Bars represent mean of measurements from three experiments. *Statistical significance from NC mice set a priori at p < 0.05. Data depicted represent two independent experiment sets.
and spleen, respectively) in blood lymphocytes the third day after irradiation (Fig. 2C). In general, a significant increase in these genes was expressed primarily on the third day after irradiation, rather than the first day after irradiation. Protein expression of radiation-responsive genes in blood lymphocytes of whole body or locally irradiated mice The detection of protein is better for the development of biomarkers than mRNA. Therefore, this study examined whether a relationship existed between mRNA expression and protein expression. One Gray of whole body or local irradiation significantly increased the protein levels of the platelet membrane glycoprotein IIb, sialyltransferase, and Cu/ZnSOD in each organ at 1 or 3 days after irradiation (Fig. 3A, B). In addition, the effects were more pronounced for local irradiation. The sialyltransferase antibody sometimes detected both the insoluble (48 kDa) and soluble (37–42 kDa) forms, and irradiation increased induction of the soluble form. The protein level of protein tyrosine kinase
was not detected because no antibody is commercially available. The protein expression patterns after 0.5, 1, and 2 Gy of radiation were examined at the third day of radiation to determine whether the protein expression after local or whole body irradiation was dependent on the radiation dose. We chose the third day because the effects were more evident than on the first day. The protein expression level in the blood lymphocytes after local irradiation to the thorax, abdomen, and left subphrenic area was increased; however, local thoracic irradiation significantly increased only platelet membrane glycoprotein IIb and Cu/ZnSOD as a function of dose when analyzed with a linear regression model. Sialyltransferase expression was also dose dependent but only when exposed to the left subphrenic area (Fig. 3C and Table 1). DISCUSSION We anticipate continued development of new technologies and procedures in the detection of radiation exposure. Moreover, these developments will continue to fuel the increased
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Fig. 2. mRNA expression of protein tyrosine kinase, platelet membrane glycoprotein IIb, sialyltransferase, and Cu/ZnSOD in peripheral blood lymphocytes or organs exposed to localized irradiation (IR). (A) Reverse transcriptase-polymerase chain reaction (RT-PCR) analysis of four genes in intestine, lung, and spleen tissue from 3 mice exposed to local 1 Gy IR and compared with gene expression in brain 3 days after IR. (B) Real-time RT-PCR or quantification for real-time RT-PCR. (C) Analysis for four genes also performed in peripheral blood lymphocytes after 1 Gy of local IR to thorax, abdomen, and left subphrenic area. Peripheral blood lymphocytes from 3 mice pooled for each RNA isolation. Bars represent mean of measurements from three experiments. Mean standard deviation calculated as ratio of nonirradiated normal control (NC) mice. *Statistical significance from NC mice set a priori at p < 0.05. Representative data of two independent experiment sets shown. Figure is continued on next page.
use of radiation technology in industry and medical diagnosis and therapy. From the experience with radiation accidents, it is evident that biologic indicators are needed to obtain enough information of the distribution and extent of radiation exposure. These biologic indicators could have an additional advantage because the radiation damage to an individual could be measured, including the variability of an individual’s radiosensitivity. Of the many biologic parameters that have been studied, chromosomal damage in lymphocytes is the most promising (24–26). Different cytogenetic changes (i.e., increased frequency of chromosomal aberrations and micronuclei), are well known in individuals exposed to ionizing radiation (27–30). However, this method is time consuming, and the recently developed large-scale gene expression showed that the individual prognosis after
radiation exposure could be estimated by determining their function. The present study has identified candidate biomarkers for radiation exposure that could be detected in blood samples of whole body or locally irradiated mice. In a previous study, five organs, including the intestine, spleen, lung, heart, and brain, were studied after 0.2 Gy of radiation exposure. RTPCR was applied to the 23 genes that have been identified by cDNA microarray analysis to show increased expression in the peripheral blood lymphocytes after 1 Gy of radiation (16). Of these, the platelet membrane glycoprotein IIb, protein tyrosine kinase, sialyltransferase, and Cu/ZnSOD were expressed in the intestine, lung, or spleen but not in the heart or brain (22), suggesting these as possible gene candidates for measuring exposure to the spleen, lung, or intestines for these
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Fig. 2. (Continued)
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Fig. 3. Protein expression in organ tissues or peripheral blood lymphocytes exposed to whole body or local irradiation (IR). After mice were exposed to 1 Gy of (A) whole body or (B) local IR, protein expression of platelet membrane glycoprotein IIb, sialyltransferase, and Cu/ZnSOD in intestine, lung, and spleen were examined at 1 and 3 days after IR. Peripheral blood lymphocytes or organs from 3 mice pooled for RNA isolation. Bars depict results from three experiments. Mean standard deviation calculated as ratio of non-IR normal control (NC) mice. *Statistical significance from NC mice set a priori at p < 0.05. Representative data of two independent experiment sets shown. (C) Ratio of peripheral blood lymphocytes isolated at 3 days after local IR to thorax, abdomen, and left subphrenic area. Pattern of protein expression of platelet membrane glycoprotein IIb, sialyltransferase, and Cu/ZnSOD examined, and peripheral blood lymphocytes from 3 mice pooled for each protein extraction, and two experiments performed for each dose. Representative data depicted as two independent experimental data sets. Figure is continued on next page.
radiation-sensitive organs. Sialyltransferase is a glycosphingolipid synthase that undergoes characteristic changes during an oncogenic transformation (30) and has influenza virus sensitivity (31). Cu/ZnSOD is a protein tyrosine kinase and platelet membrane glycoprotein IIb has functions that are more related to cellular resistance than to cell death, suggesting that the radiation exposure biomarker might not be related to function. When mRNA expression of the platelet membrane glycoprotein IIb, protein tyrosine kinase, sialyltransferase, and Cu/ZnSOD was examined in the blood after whole body irradiation, 1 Gy of radiation was found to induce these genes (Fig. 1). Similarly, local irradiation to the thorax, abdomen, or left subphrenic area also increased the mRNA levels of these genes in the irradiated lung, spleen, and intestine and blood samples (Fig. 2), suggesting that these genes might be candidates for biomarkers of radiation exposure. The most important finding of this study was the identification of possible protein biomarkers for radiation exposure, because proteins would be more suitable as biomarkers. Moreover, the changes in the expression levels of the proteins
were more pronounced compared with the changes in mRNA expression in the organs of locally irradiated mice. Therefore, the radiation induced post-translational modification of these proteins or the protein itself is more sensitive to the detection of radiation exposure. However, radiation did not induce the expression of these proteins in the blood lymphocytes after whole body irradiation. Rather, whole body irradiation reduced the protein expression (data not shown), suggesting that some proteolytic degradation in blood samples might be activated by radiation. When the protein expression in the blood lymphocytes of the locally irradiated mice was examined, no proteolytic degradation of these proteins could be detected, suggesting that local irradiation has less effect on the proteolytic degradation pathways in blood lymphocytes (the blood might be more affected by proteolytic degradation after radiation). Therefore, mRNA detection in blood samples appears to be more suitable after whole body radiation for which the detection of these proteins failed as biomarkers. Despite this, protein expression appears to be more suitable in the case of local irradiation (especially in lung tissue).
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Fig. 3. (Continued)
Table 1. Regression analysis of radiation dose dependence for protein expressions in peripheral blood lymphocytes after 1 Gy of local irradiation Thorax
Abdomen
Left subphrenic area
Irradiated region
b1*
p
b2*
p
b3*
p
Platelet membrane glycoprotein IIb Cu/ZnSOD Sialyltransferase
0.863
0.004
0.355
0.623
0.038
0.812
0.454 0.470
0.001 0.060
0.350 0.716
0.053 0.072
0.305 2.108
0.074 0.029
* Regression coefficient.
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We do not know exactly why the genes of platelet membrane glycoprotein IIb, protein tyrosine kinase, sialyltransferase, and Cu/ZnSOD could be detected in the blood lymphocytes when locally irradiated at the thorax, abdomen, and left subphrenic area. One possibility is that the lymphocytes inside the local irradiated organs might have been affected by the radiation and they were detected in the blood. Another possibility is that cytokines and soluble factors might be secreted as a result of radiation in locally irradiated organs and they, in turn, might affect the lymphocytes. Gene changes in the lymphocytes were more pronounced at 3 days (as opposed to 1 day) of radiation exposure, suggesting that the gene expression of lymphocytes after local irradiation might be an indirect consequence of radiation exposure. One of the important features of biomarkers is dose-dependent expression. Therefore, several radiation doses were used to determine whether the change in the expression of these proteins is dependent. Local radiation doses of 0.5, 1, and 2
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Gy to the thorax, abdomen, and left subphrenic area induced protein expression of platelet membrane glycoprotein IIb, sialyltransferase, and Cu/ZnSOD in blood lymphocytes; however, dose dependency was only observed for platelet membrane glycoprotein IIb and Cu/ZnSOD after thoracic irradiation and sialyltransferase after irradiation to the left subphrenic area. These results suggest possible candidates for biodosimetry, because even the 0.5-Gy radiation dose induced protein expression. In addition, it is possible that these proteins could also be candidate markers for low-dose local radiation to the thorax, abdomen, or left subphrenic area. Moreover, these proteins might be valuable biomarkers for predicting early and mechanism-specific radiation exposure, in addition to revealing the future effect of radiation earlier than with conventional methods (particularly after local radiation exposure), even though more detailed experiments are needed for the establishment of these proteins as biomarkers of radiation exposure.
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